Stem cells are known to have the remarkable ability to develop into many different cell types of a living body, such as a human or animal, during the early life and growth of the cells. Stem cells also serve as internal repair systems for many organ tissues, and serve to replenish other cells, as long as the organ-carrier (e.g., humans, animals) is still alive.
The combination of the use of stem cells with polymeric scaffolds is a promising strategy for tissue engineering and for cellular delivery. With respect to tissue engineering applications, several types of scaffolds have been used in combination with stem cells. Such scaffold materials are generally classified as natural or synthetic, with each having distinct advantages and drawbacks.
Several natural or biocompatible synthetic materials have been developed for specific scaffold applications. For example, many scaffold materials are based on proteins, such as fibrin and silk; polysaccharides, such as agarose and alginate; polymers, such as PEG (polyethylene glycol); peptides; or ceramic materials. Due to their chemical nature, these scaffold materials have been studied for use in forming bone, cartilage, heart, nerve, retinal, and vasculature tissue structures. Furthermore, these scaffold materials can be utilized in the directed differentiation of stem cells into mature phenotypes or can be used for the expansion of undifferentiated stem cells.
However, none of these developments in scaffold technology have resulted in a smart responsive scaffold (SRS) or biocompatible scaffold that can, by choice, respond to a variety of external stimuli, such as temperature, applied fields (e.g., electric field, magnetic field), surface alignment, or mechanical deformation (i.e., stress/strain) with a macroscopic ordering event (e.g., an increase in order).
The advantages of using cell delivery for therapeutic applications are significant. However, it is the methods of administration that are an important key for the success of the treatments sought, such as injecting the dispersion of cells directly into injured sites. To that end, various approaches have also been used to encapsulate cells in polymer matrices for delivery to an injured site. For example, hydrogels were thought to be attractive due to their potential in maintaining cell viability. Unfortunately, they do not offer the necessary mechanical support. Other approaches have focused on the use of porous beads for the purpose of cell delivery. In this approach, beads are cell-loaded and cultured in a medium prior to being placed or injected directly into the affected or diseased site. While beads and hydrogels may also contain bioactive agents (e.g., growth factors, proteins) they do not guide cells to differentiate into a particular cell lineage. Furthermore, because extreme care should be taken in the choice of cells to be implanted, it is of paramount importance to ensure that cells will differentiate into the desirable lineage by ‘copying’ surrounding cells. If there is heterogeneity of cells at the sites sought for repair, it will ultimately be difficult to predict that the cells will respond as desired. In addition, some scaffolds made of natural polymers (e.g., fibrin or collagen) have difficulty when used in the area of tissue regeneration because of necrosis (i.e., cell death) found at the center of these scaffolds. As a result, such scaffolds provide diminished mechanical properties and instability, which is undesirable.
Elastomers, however, have gained considerable attention for their use in forming scaffold because they offer many advantages over tough, rigid polymers. Most importantly, the physical properties of elastomers can be tuned in a way that they can withstand applied mechanical forces, such as strain, stress, and impacts, because they are soft and deformable. In addition, elastomers have been found to be suitable as carriers for drug delivery applications. Biodegradable elastomers have been mainly made of two types: thermoplastics and thermosets. Thermoplastics are easily made, but degrade heterogeneously due to the presence of crystalline and amorphous regions within the material. This leads to a rapid loss of mechanical strength. While thermosets are not as easily prepared, they offer more uniform biodegradation rates, better mechanical properties, and improved chemical resistance.
Accordingly, smart responsive scaffolds (SRSs) that respond to external stimuli resulting in a macroscopic ordering event have been developed by at least one or more inventors in this application and are disclosed in PCT Patent Application Publication No. WO 2014/172261, the disclosure of which is incorporated herein by reference. These unique, smart responsive scaffolds (SRSs) utilize thermoset elastomers that are based on three-arm star block copolymers (SBCs) using ring opening polymerization of suitable monomers followed by cross-linking to form the elastomer. Since their discovery, these biocompatible and biodegradable elastomers have become an important factor in the fabrication of modern biomedical technologies, such as tissue engineering and drug delivery. Their elastomeric characteristics not only mimic the mechanical deformation of the biological supporting matrix, but also facilitate cell interaction by modulating cellular behavior. Among the biodegradable elastomeric soft materials that have been developed, the polycaprolactone (PCL)-Polylactide acid (PLA)-based elastomers have become ideal for use in these biomedical applications due to their known biocompatibility and biodegradability. The star-shaped block copolymer (SBC) of PCL with other biocompatible blocks, such as Polylactide (PLA), offer multiple polymer arms for better controlled elasticity and degradability. PCL-based scaffolds are well known and are one of the most commonly used materials for such new biomedical applications.
Unfortunately, while the new three-arm star block copolymer with liquid crystal side chain-based elastomeric films have shown promise in targeting on aligned cell culturing with the guidance of liquid crystal behaviors, there are limitations on the elastomeric films' ability to maximize the performance of the elastomeric scaffold for better cell culturing applications. That is, the performance of the material as an elastomeric scaffold remains limited by any of a number of issues, including the composition of the star block copolymers, the molecular structure of the cross-link sites and morphology of the scaffold itself.
For example, the current smart responsive scaffolds (SRSs), while porous, do not contain any interconnecting pores. That is, the pores of these three-arm star block copolymer-based scaffold materials having pendant liquid crystal side chains do not develop well-defined, interconnected pores, and instead, only have pores that provide for additional surface area for cell migration, but do not allow for a well-defined porosity with interconnected pores that will allow for better tissue development and improved cell adherence and growth. It is well known in the art that, in order to effectively utilize the elastomer-based scaffolds with liquid crystal side chains for tissue engineering and biomedical applications, the scaffolds must have well-defined porosity and surface properties that provide support for cell adherence, growth, and mass transport of the nutrients/waste in and out of the scaffold pores under physiological conditions.
There are at least three types of mass transport to be considered for three-dimensional (3D) cell cultures: (a) oxygen mass transport (i.e., controlling metabolism rate), (b) mass transport of nutrients to cells, and (c) waste mass transport (i.e., eliminating toxins that, for example, raise pH to toxic levels, among others). Just as important, controlled porosity of the elastomer promotes 3D cell-elastomer interactions, space for extracellular matrix (ECM) formation and the possibility of linking molecular entities to allow binding of cell growth factors or other proteins to enhance cellular adhesion and ECM formation.
Chemical and photo-initiated cross-linking of linear polymer precursors or multi-arm polyester precursors, also known as star shaped copolymers, have been used as methods for designing porous polymeric materials for biomedical applications, but these methods only provide pores and do not actively promote controlled porosity or interconnecting pores. Such methods for the preparation of porous elastomers are rare. One example includes the incorporation of a leachable solid, such as paraffin beads, into a polymer and draining it after forming porous elastomer materials. Other methods include the use of a solvent vapor annealing method developed to prepare thin films of block copolymers with well-defined porosity.
Despite many advantages, these methods still have certain limitations. For example, photo-initiated cross-linking is limited to polyesters and requires an extra step to attach an unsaturated moiety to polymers prior to cross-linking. This is both time consuming and costly.
Thus, instead of fabricating elastomer films, developing liquid crystal elastomeric foams for high-efficiency cell culture offers the promise of higher efficiency and new tissue engineering functions. These scaffold foams will enable the mass transport of oxygen and nutrients important to the development of healthy cells as well as enable the rapid flushing of wastes from the scaffold to prevent degradation of the cell culture.
Fabrication of porous (i.e., containing pores) elastomer scaffolds has been previously attempted, and various methods have been pursued. For example, porous foam has been made using a micelle template, but the pore size is usually limited to only a few microns due to the size of the micelles used. Other scaffolds have been built up with microfibers generated by electrospun templates, or gas bubble templates, but these do not provide for adequate control and consistency of the porosity.
Thus, the need exists for alternative 3D scaffolds that provide suitable pores sizes that are both controlled and consistent. For some applications, hollowed, channeled scaffolds are desired for mimicking vascular conduits within a body. However, most of these hollowed channel fabrications rely on lithography and require time-consuming and equipment-demanding experiment conditions. Furthermore, many of these channeled scaffolds are only simplified models of the vascular conduits in vivo, consisting of aligned channel arrays due to their relatively-easy fabrication. The need to produce more complex networks and scaffolds having interconnected pores in the range of between 100 microns and 500 microns, and/or micro-channels of similar, or slightly larger, size in a simple and convenient way continues to exist.